Mutational and gene expression analysis of mtrDEF, omcA and mtrCAB during arsenate and iron reduction in Shewanella sp. ANA-3.

Arsenate respiration and Fe(III) reduction are important processes that influence the fate and transport of arsenic in the environment. The goal of this study was to investigate the impact of arsenate on Fe(III) reduction using arsenate and Fe(III) reduction deficient mutants of Shewanella sp. strain ANA-3. Ferrihydrite reduction in the absence of arsenate was similar for an arsenate reduction mutant (arrA and arsC deletion strain of ANA-3) compared with wild-type ANA-3. However, the presence of arsenate adsorbed onto ferrihydrite impeded Fe(III) reduction for the arsenate reduction mutant but not in the wild-type. In an Fe(III) reduction mutant (mtrDEF, omcA, mtrCAB null mutant of ANA-3), arsenate was reduced similarly to wild-type ANA-3 indicating the Fe(III) reduction pathway is not required for ferrihydrite-associated arsenate reduction. Expression analysis of the mtr/omc gene cluster of ANA-3 showed that omcA and mtrCAB were expressed under soluble Fe(III), ferrihydrite and arsenate growth conditions and not in aerobically grown cells. Expression of arrA was greater with ferrihydrite pre-adsorbed with arsenate relative to ferrihydrite only. Lastly, arrA and mtrA were simultaneously induced in cells shifted to anaerobic conditions and exposed to soluble Fe(III) and arsenate. These observations suggest that, unlike Fe(III), arsenate can co-induce operons (arr and mtr) implicated in arsenic mobilization.

The ArsR repressor mediates arsenite-dependent regulation of arsenate respiration and detoxification operons of Shewanella sp. strain ANA-3.

Microbial arsenate reduction affects the fate and transport of arsenic in the environment. Arsenate respiratory (arr) and detoxifying (ars) reduction pathways in Shewanella sp. strain ANA-3 are induced by arsenite and under anaerobic conditions. Here it is shown that an ArsR family protein, called ArsR2, regulates the arsenate respiratory reduction pathway in response to elevated arsenite under anaerobic conditions. Strains lacking arsR2 grew faster in the presence of high levels of arsenite (3 mM). Moreover, expression of arrA and arsC (arsenate reductase-encoding genes) in the DeltaarsR2 mutant of ANA-3 were increased in cells grown under anaerobic conditions and in the absence of arsenic. Mutations in putative arsenic binding amino acid residues in ArsR2 (substitutions of Cys-30 and Cys-32 with Ser) resulted in ANA-3 strains that exhibited anaerobic growth deficiencies with high levels of arsenite and arsenate. DNA binding studies with purified ArsR2 showed that ArsR2 binding to the arr promoter region was impaired by trivalent arsenicals such as arsenite and phenylarsine oxide. However, ArsR2 binding occurred in the presence of arsenate. A second known regulator of the arr operon, cyclic AMP (cAMP)-cAMP receptor protein (CRP), could bind simultaneously with ArsR2 within the arr promoter region. It is concluded that ArsR2 is most likely the major arsenite-dependent regulator of arr and ars operons in Shewanella sp. strain ANA-3. However, anaerobic growth on arsenate will require coregulation with global regulators such as cAMP-CRP.

Functional roles of arcA, etrA, cyclic AMP (cAMP)-cAMP receptor protein, and cya in the arsenate respiration pathway in Shewanella sp. strain ANA-3.

Microbial arsenate respiration can enhance arsenic release from arsenic-bearing minerals--a process that can cause arsenic contamination of water. In Shewanella sp. strain ANA-3, the arsenate respiration genes (arrAB) are induced under anaerobic conditions with arsenate and arsenite. Here we report how genes that encode anaerobic regulator (arcA and etrA [fnr homolog]) and carbon catabolite repression (crp and cya) proteins affect arsenate respiration in ANA-3. Transcription of arcA, etrA, and crp in ANA-3 was similar in cells grown on arsenate and cells grown under aerobic conditions. ANA-3 strains lacking arcA and etrA showed minor to moderate growth defects, respectively, with arsenate. However, crp was essential for growth on arsenate. In contrast to the wild-type strain, arrA was not induced in the crp mutant in cultures shifted from aerobic to anaerobic conditions containing arsenate. This indicated that cyclic AMP (cAMP)-cyclic AMP receptor (CRP) activates arr operon transcription. Computation analysis for genome-wide CRP binding motifs identified a putative binding motif within the arr promoter region. This was verified by electrophoretic mobility shift assays with cAMP-CRP and several DNA probes. Lastly, four putative adenylate cyclase (cya) genes were identified in the genome. One particular cya-like gene was differentially expressed under aerobic versus arsenate respiration conditions. Moreover, a double mutant lacking two of the cya-like genes could not grow with arsenate as a terminal electron acceptor; exogenous cAMP could complement growth of the double cya mutant. It is concluded that the components of the carbon catabolite repression system are essential to regulating arsenate respiratory reduction in Shewanella sp. strain ANA-3.

Complete genome sequence of Pyrobaculum oguniense.

Pyrobaculum oguniense TE7 is an aerobic hyperthermophilic crenarchaeon isolated from a hot spring in Japan. Here we describe its main chromosome of 2,436,033 bp, with three large-scale inversions and an extra-chromosomal element of 16,887 bp. We have annotated 2,800 protein-coding genes and 145 RNA genes in this genome, including nine H/ACA-like small RNA, 83 predicted C/D box small RNA, and 47 transfer RNA genes. Comparative analyses with the closest known relative, the anaerobe Pyrobaculum arsenaticum from Italy, reveals unexpectedly high synteny and nucleotide identity between these two geographically distant species. Deep sequencing of a mixture of genomic DNA from multiple cells has illuminated some of the genome dynamics potentially shared with other species in this genus.

The cymA gene, encoding a tetraheme c-type cytochrome, is required for arsenate respiration in Shewanella species.

In Shewanella sp. strain ANA-3, utilization of arsenate as a terminal electron acceptor is conferred by a two-gene operon, arrAB, which lacks a gene encoding a membrane-anchoring subunit for the soluble ArrAB protein complex. Analysis of the genome sequence of Shewanella putrefaciens strain CN-32 showed that it also contained the same arrAB operon with 100% nucleotide identity. Here, we report that CN-32 respires arsenate and that this metabolism is dependent on arrA and an additional gene encoding a membrane-associated tetraheme c-type cytochrome, cymA. Deletion of cymA in ANA-3 also eliminated growth on and reduction of arsenate. The DeltacymA strains of CN-32 and ANA-3 negatively affected the reduction of Fe(III) and Mn(IV) but not growth on nitrate. Unlike the CN-32 DeltacymA strain, growth on fumarate was absent in the DeltacymA strain of ANA-3. Both homologous and heterologous complementation of cymA in trans restored growth on arsenate in DeltacymA strains of both CN-32 and ANA-3. Transcription patterns of cymA showed that it was induced under anaerobic conditions in the presence of fumarate and arsenate. Nitrate-grown cells exhibited the greatest level of cymA expression in both wild-type strains. Lastly, site-directed mutagenesis of the first Cys to Ser in each of the four CXXCH c-heme binding motifs of the CN-32 CymA nearly eliminated growth on and reduction of arsenate. Together, these results indicate that the biochemical mechanism of arsenate respiration and reduction requires the interactions of ArrAB with a membrane-associated tetraheme cytochrome, which in the non-arsenate-respiring Shewanella species Shewanella oneidensis strain MR-1, has pleiotropic effects on Fe(III), Mn(IV), dimethyl sulfoxide, nitrate, nitrite, and fumarate respiration.